Asparaginase is a standard and critical component in the therapy of childhood acute lymphoblastic leukemia. Asparagine synthetase (ASNS) and the basic region leucine zipper activating transcription factor 5 (ATF5) and arginosuccinate synthase 1 (ASS1) have been shown to mediate the antileukemic effect of asparaginase and to display variable expression between leukemia cells that are resistant and sensitive to treatment. Fourteen polymorphisms in the regulatory and coding regions of these genes were investigated for an association with acute lymphoblastic leukemia outcome. Lower event-free survival (EFS) was associated with ATF5 T1562C, tandem-repeat ASNS polymorphism, derived haplotype, and ASS1 G1343T and G34T substitutions (P ≤ .03). Associations were limited to patients who received Escherichia coli asparaginase. Variations that sustained correction for multiple testing (ATF5 T1562C, P = .005; ASNS tandem-repeat and related haplotype, P ≤ .01) were subsequently analyzed in the replication cohort. The E coli-dependent association of the ATF5 T1562 allele with reduced EFS was confirmed (P = .01). A gene-reporter assay showed that the haplotype tagged by T1562 had higher promoter activity (P ≤ .01). The remaining regulatory polymorphisms also appeared to affect ATF5 function; 2 additional high-activity haplotypes were identified (P ≤ .02) and were further corroborated by quantitative mRNA analysis in lymphoblastoid cell lines. The ATF5-regulated increase in ASNS expression in response to more efficacious E coli-induced asparagine depletion may explain our observed results.